The major goal of our research is to understand how proteins specifically recognize DNA and how this interaction modulates transcriptional regulation. Heat shock transcription factor (HSF) has been chosen for study because of its important biological role, as well as its unique structure. HSF is the transcriptional activator for the heat shock response, an evolutionarily conserved mechanism which protects cells from heat and other environmental and chemical stimuli. HSF is found in all eukaryotes, from yeast to humans. HSF encompasses a new family of proteins, with a highly conserved DNA binding domain and a novel trimerization domain. The recent structure of the DNA binding domain shows that it has a topology similar to helix proteins such as catabolite activator protein (CAP) and histone H5. Despite this similarity, HSF differs from the classical helix-turn-helix motif: instead of the three amino acid turn between the two recognition helices, HSF has a loop of five amino acids, which causes a major change in the angle between the two helices. HSF may constitute a 'sub-class" of the helix-turn-helix motif. The trimerization domain includes a triple- stranded alpha-helical coiled-coil. HSF is the only known example of a trimeric DNA binding protein, leading to interesting questions about how it binds to DNA. The key to studying a protein of the size and complexity of HSF is to obtain smaller fragments that are amenable to physical studies. The specific aims of this proposal focus on a study of the DNA binding and trimerization domains, and they include: characterization of the biochemical and physical behavior of a trimeric DNA binding protein, continuation of the successful crystallization studies, and utilization of genetics as a complementary tool to the structural and physical characterization of these domains. These studies will lead to the long term goal of understanding how a trimeric protein like HSF can interact with DNA.